An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. As mentioned in the last report, we have prepared gold self-assembled nanospheres and quantum dots containing sugar derivative and reported preliminary details on their function. The in vivo experiments with our gold nanospheres in mice were conflicting, so we retreated to basics and performed more rigorous characterization and explored a host of new syntheses that allowed for production of more uniform particles. We proeeded to systematically study the optimum procedure, from several related methods, that offered the highest quality particles with regards to stability and uniformity. We are still examining these data in various media to test for stability. We have prepared the TF antigen in different contexts (attached to both serine and threonine) and linked them to particles. Our TF particles have now been shown in pull down experiments to bind to Galectin-3 and integrin complexes related to metastasis. We put a heavy emphasis on preparing particles that encompassed what we consider the best antigen, a glycopeptide from tumor associated cell-surface mucins, and combined that with various concentrations of linker and T-helper epitope to construct particles that may act as novel immunogens. We prepared at least seven separate particles with various placements of the disaccharide on the peptide, and along with linker and a 28-residue portion of C3d, a domain of complement component 3, which has been shown to enhance the immune response to T-cell independent epitopes like carbohydrates. These particles were injected into mice and the sera were analyzed for immune responses. So far, it looks like the particles elicit a response and we are now examining their specificity. We are exteremely excited about this since it could be te discovery of a completely new way of delivering and presenting tumor antigen and also adjust the paradigm of the need for covalent attachment of B- and T-cell epitopes in the same molecule. We also have explored other methods for synthesizing TF antigen on peptides. An enzymatic protocol has been worked out to add the first sugar and we have a procedure for preparing the second glycosyl transferase to add the second sugar. We have explores different peptide lengths and find that as the length increases, so does the number of sugars that get transferred to the serines and threonines of the peptide backbone